The overall control of iron homeostasis occurs at the transport step in the epithelium of the proximal small bowel, where absorption is precisely regulated to match body iron losses. Importantly, perturbations in intestinal iron transport are associated with several important disease states in humans, including anemia of chronic disease and hemochromatosis. Intestinal copper transport is enhanced in rats during iron-deficiency, and this is likely a physiological response related to the role of dietary copper in various aspects of overall body iron homeostasis. Interestingly, the Menkes copper ATPase (Atpya) is strongly induced in the duodenal mucosa of iron-deprived rats at different postnatal ages along with Divalent Metal Transporter i (Dmti), which can transport iron and copper. Thus, the overall goals of this proposal are i) to determine the roles that Dmti and Atpya play in the induction of copper transport during iron-deprivation, 2) to decipher the molecular mechanisms of induction of Dmti and Atpya during iron-deprivation and 3) to determine the effect that copper has on molecular mechanisms of trans-epithelial iron transport in the intestine. This will be accomplished by utilizing cell culture and rodent models of intestinal iron transport. Specific AIM i will test the hypothesis that induction of Dmti and Atpya is responsible for increased transepithelial copper transport seen during iron-deprivation. Iron and copper transport studies and siRNA knockdowns will be performed in our in vitro model of the intestinal epithelium, the IEC-6 cells. Once the transporter(s) involved in the induction of copper transport during iron-deficiency have been identified, we will perform complementary studies in in vivo models of iron-deficiency, including wild-type, iron-deficient rats, Belgrade (i.e. Dmti-deficient) rats and Atpya knockout mice. Specific AIM 2, will test the hypothesis that Dmti and Atpya are regulated by distinct molecular mechanisms during iron-deficiency. Iron-dependent, post- transcriptional regulation of Dmti will be examined (mediated by the 3'IRE) and transcriptional regulation of Atpya in IEC-6 cells will be studied. Finally, specific AIM 3 will test the hypothesis that increased copper transport during iron deficiency functions to enhance copper-dependent aspects of intestinal iron absorption. To accomplish this goal, iron transport studies will be performed in membrane vesicles isolated from iron-deficient rats deprived of dietary copper, and hephaestin activity in enterocytes and ceruloplasmin activity in serum will be determined. Overall, these studies will allow further definition of the copper- dependent processes that are involved in enhancing intestinal iron absorption during states of iron- deficiency. A detailed understanding of these relationships is critical, as intestinal iron transport controls overall body iron homeostasis. Moreover, this proposed investigation is novel, as studies addressing the impact of increased enterocyte and liver copper levels during iron-deficiency have not been reported to date.